QPOLE: A Quick, Simple, and Cheap Alternative for POLE Sequencing in Endometrial Cancer by Multiplex Genotyping Quantitative Polymerase Chain Reaction

Abstract

PURPOSE Detection of 11 pathogenic variants in the POLE gene in endometrial cancer (EC) is critically important to identify women with a good prognosis and reduce overtreatment. Currently, POLE status is determined by DNA sequencing, which can be expensive, relatively time-consuming, and unavailable in hospitals without specialized equipment and personnel. This may hamper the implementation of POLE-testing in clinical practice. To overcome this, we developed and validated a rapid, low-cost POLE hotspot test by a quantitative polymerase chain reaction (qPCR) assay, QPOLE. MATERIALS AND METHODS Primer and fluorescence-labeled 5′-nuclease probe sequences of the 11 established pathogenic POLE mutations were designed. Three assays, QPOLE-frequent for the most common mutations and QPOLE-rare-1 and QPOLE-rare-2 for the rare variants, were developed and optimized using DNA extracted from formalin-fixed paraffin-embedded tumor tissues. The simplicity of the design enables POLE status assessment within 4-6 hours after DNA isolation. An interlaboratory external validation study was performed to determine the practical feasibility of this assay. RESULTS Cutoffs for POLE wild-type, POLE-mutant, equivocal, and failed results were predefined on the basis of a subset of POLE mutants and POLE wild-types for the internal and external validation. For equivocal cases, additional DNA sequencing is recommended. Performance in 282 EC cases, of which 99 were POLE-mutated, demonstrated an overall accuracy of 98.6% (95% CI, 97.2 to 99.9), a sensitivity of 95.2% (95% CI, 90.7 to 99.8), and a specificity of 100%. After DNA sequencing of 8.8% equivocal cases, the final sensitivity and specificity were 96.0% (95% CI, 92.1 to 99.8) and 100%. External validation confirmed feasibility and accuracy. CONCLUSION QPOLE is a qPCR assay that is a quick, simple, and reliable alternative for DNA sequencing. QPOLE detects all pathogenic variants in the exonuclease domain of the POLE gene. QPOLE will make low-cost POLE-testing available for all women with EC around the globe.

Molecular Classification of Endometrial Cancer and Its Impact on Therapy Selection

“…Tian et al showed that P286R and V411L variants are associated with higher a Pol ε activity than POLEwt [90]. [23,24,86,92]. Sanger sequencing preferentially amplifies the normal sequence of the POLE gene.…”

“…However, during the analysis of 359 cases, McAlpine et al identified most (82%) POLE mutations as being pathogenic [ 85 ]. The most frequent pathogenic mutations of POLE are located in 11 loci across 9, 11, 13, and 14 exons [ 86 ]. Among them, substitutions P286R, V411L, S297F, A456P, and S459F were indicated by León-Castillo et al as being the most commonly occurring, while mutations in the six remaining domains (i.e., M295R, F367S, D368Y, L424I, P436R, and M444K) were rarer [ 86 , 87 ] ( Table 1 ).…”

“…Currently, POLE mutations are mostly determined by methods based on DNA sequencing, such as Sanger and next-generation sequencing [ 23 , 24 , 86 , 92 ]. Sanger sequencing preferentially amplifies the normal sequence of the POLE gene.…”

Molecular Classification of Endometrial Cancer and the 2023 FIGO Staging: Exploring the Challenges and Opportunities for Pathologists

“…To overcome this, Van den Heerik et al crafted a solution to this issue by developing QPOLE, a POLE hotspot test that is both quick, taking approximately 2 h, and cost-effective. The test uses a quantitative polymerase chain reaction (qPCR) assay [ 68 ]. This methodology stands out as an efficient and accurate testing procedure for POLE in endometrial biopsy and hysterectomy specimens.…”

Moving into the modern era of molecular classification for endometrial cancer

“…14,24,25 Other groups have published on cost-effective and efficient approaches for POLE sequencing, which can be utilized in the molecular work-up of ECs. [26][27][28][29] These F I G U R E 1 Schematic of the molecular subtype classification of endometrial carcinoma using surrogates based on immunohistochemistry and/or molecular data. The distribution of histological types by molecular subtype are obtained from Rios-Doria et al 14 Abn, abnormal; CN-H, copy number-high; CN-L, copy number-low; ED, exonuclease domain; IHC, immunohistochemistry; MMR, mismatch repair; MSI-H, microsatellite instability-high; NGS, next generation sequencing; NSMP, no specific molecular profile; POLE, polymerase epsilon.…”